CD8 epitope from HIV-1 protein Vpu

ABSTRACT

The invention relates to the identification and the selection of CTL epitopes able to induce a protection against an HIV infection. More particularly, the invention is concerned with peptides and nucleic acid sequence coding for these peptides derived from HIV-1 proteins such as GAG, POL, ENV, VIF, TAT, VPU, REV and their applications. Preferably the immunogenic peptides are selected from the group consisting of SEQ ID NOs:1 to 18 and functional derivatives thereof. The invention also relates to antibodies directed against said peptides.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national phase application based onPCT/IB02/04576, filed Sep. 27, 2002, the content of which isincorporated herein by reference.

The present invention relates to the identification and the selection ofCTL epitopes able to induce a protection against an HIV infection. Moreparticularly, the invention is concerned with peptides and nucleic acidsequences coding for these peptides derived from HIV-1 proteins such asGAG, POL, ENV, VIF, TAT, VPU, REV.

Many scientific publications disclosed HIV epitopes having the propertyto induce in animals a B cell response as well as a T-cell responsespecific for HIV. The importance of CTL response under HIV control havebeen demonstrated in many experiences. Indeed, it is known that there isan inverse correlation between CTL response and HIV viremia in patients;that CD8 (CTL) depletion in monkeys leads to an increase of the viremia;that there are subjects who are continually exposed to the virus but arenot infected and who possess a strong CTL response and that specific HIVCTL clones have been shown to inhibit the viral replication in vitro.

Although that numerous HIV-1 proteins are known and under study (see WO01/27291; Altfeld M. et al. (2001), The Journal of Immunology, 167:2743–2752; and Jin X., et al. (2000), AIDS research and Humanretroviruses, 16: 67–76), the extent to which these proteins aretargeted in natural infection, as well as precise CTL epitopes withinthem, remain to be defined.

Therefore, there is a strong need for peptides and epitopes that arecapable to induce in animals a B cell response, as well as a T cellresponse, against HIV-1 proteins, and to the use of such peptides andepitopes in the diagnostic, in the prevention/protection against an HIVinfection and in the treatment of HIV.

The present invention fulfils these needs and also other needs whichwill be apparent to those skilled in the art upon reading the followingspecification.

The present invention relates to the identification and the selection ofCTL epitopes able to induce a protection against an HIV infection. Moreparticularly, the invention is concerned with peptides and nucleic acidsequences coding for these peptides derived from HIV-1 proteins such asGAG, POL, ENV, VIF, TAT, VPU, REV.

More particularly, the invention provides new immunogenic peptides andepitopes capable of inducing a cytotoxic CD8 T-lymphocytes (CTLs) aslarge as possible.

The invention also provides antibodies binding to the immunogenicpeptides of the invention thereto.

The invention further relates to pharmaceutical compositions and tomethods for inducing/stimulating of an immune response into a subject.

An advantage of the present invention is that it identifies, among HIV-1proteins, epitopes capable of inducing a specific cytotoxic CD8T-lymphocytes (CTLs) response. The invention also provides new HIV CTLepitopes leading in the increase of the breadth of the HIV/CTL responseafter vaccination.

Other objects and advantages of the present invention will be apparentupon reading the following non-restrictive detailed description, madewith reference to the accompanying drawings.

FIG. 1 is a graph showing the pattern for IFNγ ELISPOT responses againstHIV-1 derived epitopic peptides (known and new “coding” peptides) of onepatient (BNC).

FIG. 2 shows an ELISPOT assay on human HIV⁺ PBMCs with <<coding>>peptides.

FIG. 3 shows an ELISPOT assay on human HIV⁺ PBMCs with the <<ARFP>>peptides.

FIG. 4 is a graph showing BNC's pattern for ⁵¹Cr release assay againstpredicted HIV-1 derived “coding” epitopic peptides.

FIG. 5 is a graph showing BNC's and TAH's pattern for ⁵¹Cr release assayagainst predicted HIV-1 derived ARFP epitopic peptides. <<ARFP>>peptides and peptides No 5, 6, 7, 8, 9, 10, 11, are issued from thesequence of the BRU strain (Wain-Hobson et al., 1985).

FIG. 6A shows IFNγ intracellular labeling with PBMCs of the patient TAH.

FIG. 6B shows IL4 intracellular labeling with TAH PBMCs.

A) DEFINITIONS

In order to provide an even clearer and more consistent understanding ofthe specification and the claims, including the scope given herein tosuch terms, the following definitions are provided:

Allelic variant: refers to a peptide having from one to two amino acidsubstitutions from a parent peptide, but retaining the bindingspecificity and/or physiological activity of the parent peptide. As usedherein, “retaining the binding specificity of the parent peptide” meansbeing able to bind to a monoclonal or polyclonal antibody that binds toone of the peptides with an affinity that is at least one-tenth, morepreferably at least one-half, and most preferably at least as great asthat of one of the actual peptides. Determination of such affinity ispreferably conducted under standard competitive binding immunoassayconditions. “Retaining the physiological activity of the parent peptide”means retaining the ability of any one of the peptides shown in SEQ IDNOs 1 to 18. The term “allelic variants” is specifically intended toinclude any human analogs of the peptides set forth in SEQ ID NOS. 1 to18, which do not have the identical amino acid sequence thereof.

Antibody: refers to a glycoprotein produced by lymphoid cells inresponse to a stimulation with an immunogen. Antibodies possess theability to react in vitro and in vivo specifically and selectively withan antigenic determinant or epitope eliciting their production or withan antigenic determinant closely related to the homologous antigen.

As used herein, a protein/peptide is said to be a “chemical derivative”of another protein/peptide when it contains additional chemical moietiesnot normally part of the protein/peptide, said moieties being added byusing techniques well known in the art. Such moieties may improve theprotein/peptide solubility, absorption, bioavailability, biological halflife, and the like. Any undesirable toxicity and side-effects of theprotein/peptide may be attenuated and even eliminated by using suchmoieties. For example, proteins/peptides can be covalently coupled tobiocompatible polymers (polyvinyl-alcohol, polyethylene-glycol, etc) inorder to improve stability or to decrease/increase their antigenicity.

Derived: A protein/peptide is said to “derive” from aprotein/peptide/gene or from a fragment thereof when suchprotein/peptide/gene comprises at least one portion, substantiallysimilar in its sequence, to the native protein/peptide/gene or to afragment thereof.

Fragment: refers to a section of a molecule, such as protein/peptide ornucleic acid, and is meant to refer to any portion of the amino acid ornucleotide sequence.

A “functional derivative”, as is generally understood and used herein,refers to a protein/peptide sequence that possesses a functionalbiological activity that is substantially similar to the biologicalactivity of the whole protein/peptide sequence. A functional derivativeof a protein/peptide may or may not contain post-translationalmodifications such as covalently linked carbohydrate, if suchmodification is not necessary for the performance of a specificfunction. The term “functional derivative” is intended to the“fragments”, “segments”, “variants”, “allelic variants”, “analogs” or“chemical derivatives” of a protein/peptide.

Fusion protein: A protein formed by the expression of a hybrid gene madeby combining two gene sequences. Typically, this is accomplished bycloning a cDNA into an expression vector in frame with an existing gene.

Immunogenic: Refers to the property of a molecule or compound, such as aprotein/peptide/nucleic acid to induce in vivo or in vitro a cellular orhumoral immune response.

Immune response: Refers to an in vivo or in vitro reaction in responseto a challenge by an immunogen. An immune response is generallyexpressed by an antibody production and/or a cell-mediated immunity orimmunologic tolerance.

Isolated or Purified: Means altered “by the hand of man” from itsnatural state, i.e., if it occurs in nature, it has been changed orremoved from its original environment, or both. For example, apolynucleotide naturally present in a living organism is not “isolated”,the same polynucleotide separated from the coexisting materials of itsnatural state, obtained by cloning, amplification and/or chemicalsynthesis is “isolated” as the term is employed herein. Moreover, apolynucleotide that is introduced into an organism by transformation,genetic manipulation or by any other recombinant method is “isolated”even if it is still present in said organism.

Oligonucleotide or Polynucleotide means nucleic acid, eitherdesoxyribonucleic acid (DNA), or ribonucleic acid (RNA), insingle-stranded or double-stranded form or molecule having onenucleotide or more, whether occurring naturally or non-naturally in aparticular cell, tissue or organism, and any chemical modificationsthereof. Such modifications include, but are not limited to providingother chemical groups that incorporate additional charge,polarizability, hydrogen bonding or electrostatic interaction to one ormore of nucleic acid bases of the oligonucleotide. Examples ofmodifications are, but are not limited to, modifying the bases such assubstitution of 5-bromouracil, 5-position pyrimidine modifications,8-position purine modifications, modifications at cytosine exocyclicamines, 2′-position sugar modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine, backbone modifications, 3′ and 5′ modifications such ascapping, and the like. Are also compatible with the current invention,modifications that occur after each round of amplification in areversible or irreversible manner.

Peptide: includes any natural or synthetic compounds containing two ormore amino acids connected to each other in a linear array by peptidesbonds. Such linear array may optionally be cyclic, i.e., the ends of thelinear peptide or the side chains of amino acids within the peptide maybe joined, e.g., by a chemical bond. The peptides according to theinvention may include from about three to about 500 amino acids, and mayfurther include secondary, tertiary or quaternary structures, as well asintermolecular associations with other peptides or other non-peptidemolecules. Such intermolecular associations may be through, withoutlimitation, covalent bonding (e.g., through disulfide linkages), orthrough chelation, electrostatic interactions, hydrophobic interactions,hydrogen bonding, ion-dipole interactions, dipoledipole interactions, orany combination of the above. This term also includes proteins andfragments thereof produced through recombinant means, and/or that hasbeen associated or not with other peptides coding for tumoral, viral,bacterial or fungic epitopes for forming a fusion protein.

Specific lysis: in the enclosed example, specific lysis means that atleast 10% of the HIV infected cells are killed within 4 hours.

Vaccine: a preparation of antigenic material comprising at least onepeptide according to the invention and/or at least one polynucleotidecoding the same, that can be used to stimulate a specific immuneresponse able to confer a protection against HIV or limit an HIVinfection.

The term “variant” as is generally understood and used herein, refers toa protein that is substantially similar in structure and biologicalactivity to either the protein or fragment thereof. Thus two proteinsare considered variants if they possess a common activity and maysubstitute each other, even if the amino acid sequence, the secondary,tertiary, or quaternary structure of one of the proteins is notidentical to that found in the other.

Vector: A self-replicating RNA or DNA molecule which can be used totransfer an RNA or DNA segment from one organism to another. Vectors areparticularly useful for manipulating genetic constructs and differentvectors may have properties particularly appropriate to expressprotein(s) in a recipient during cloning procedures and may comprisedifferent selectable markers. Bacterial plasmids are commonly usedvectors.

B) General Overview of the Invention

The present invention relates to derived peptides from HIV-1 proteins,and more particularly from the GAG, POL, ENV, VIF, TAT, VPU, REVproteins of HIV-1. The invention also relates to nucleic acid sequencescoding for said peptides. The peptides and the nucleic acids of theinvention may be useful for the prevention and/or treatment of HIV-1infections.

The peptides of the invention may also be used for detecting an earlyCTL response against of HIV-1, for priming in vitro immune cells of aeukaryotic subject, for stimulating a subject immune response, andstimulating ex vivo or in vivo a human immune response against HIV-1.

The peptides/nucleic acids of the present invention may be used in allmembers of the class Vertebrates. Preferably, the vertebrate is amammalian subject including, without limitation, human and non-humanprimates, farm animals, domestic animals, laboratory animals.

C) Immunogenic Peptides and Corresponding Nucleotides

In one aspect, the invention is directed to immunogenic peptides thatderive from the HIV-1 antigen. Advantageously, the peptides of theinvention are capable of inducing an in vitro, ex vivo and/or in vivoCTL response against HIV-1 in a mammal.

More particularly, the immunogenic peptides according to the inventioncan induce in vitro, ex vivo and/or in vivo specific cytotoxic CD8T-lymphocytes (CTLs) capable of eliminating specifically HIV-1 infectedcells. Preferably, the immunogenic peptides comprise between 9 and 30amino acid having at least 60% homology with any one of the peptide ofSEQ ID NOs 1 to 18. More preferably, the peptides of the invention arenonameric or docameric peptides and even more preferably, they areselected from the group consisting of SEQ ID NOs:1 to 18 and even morethose particularly selected from the group consisting of SEQ ID Nos: 1,2, 3, 6 and 15 (see Tables 1 and 2 hereafter). However the presentinvention is not restricted to these specific peptides by it encompassesalso “functional derivatives” thereof, including “fragments”,“variants”, “allelic variants”, “analogs” or “chemical derivatives” ofthese peptides, having a comparable specificity and/or biologicalactivity as the peptides of SEQ ID NOs:1 to 18.

Modified peptides within the scope of the present invention includethose in which one or more amide bond is replaced by a non-amide bond,and/or one or more amino acid side chain is replaced by a differentchemical moiety, or one or more of the N-terminus, the C-terminus, orone or more side chain is protected by a protecting group, and doublebonds and/or cyclization and/or stereospecificity is introduced into theamino acid chain to increase rigidity, and/or binding affinity and/orenhance resistance to enzymatic degradation, of the peptides of SEQ IDNOs:1 to 18. Since all the variations are known in the art, a personskilled in the art will be able to produce, test, identify and selectother peptides/epitopes according to the present invention (see e.g.Horwell et al, Bioorg. Med. Chem. 4: 1573 (1976); Liskamp et al., Recl.Trav. Chim. Pays-Bas 1: 113 (1994); Gante et al, Angew. Chem. Int. Ed.Engl. 33: 1699 (1994); and Seebach et al, Helv. Chim. Acta 49: 313(1996)).

For instance, it is possible to substitute amino acids by equivalentamino acids. “Equivalent amino acid” is used herein to name any aminoacid that may substituted for one of the amino acids belonging to theinitial peptide structure without modifying the hydrophilicityproperties and the biological target of the initial peptide structure.Preferably, the peptides containing one or several “equivalent” aminoacids retain their specificity and affinity properties to the biologicaltargets of the peptide according to the invention. In other words, the“equivalent” amino acids are those which allow the generation or thepreparation of a polypeptide or peptide with a modified sequence asregards to the peptides according to the invention, said modifiedpolypeptide or peptide being able to act as an agonist or an antagonistmolecule of the peptide according to the invention. These equivalentamino acids may be determined by their structural homology with theinitial amino acids to be replaced and by their biological activity onthe target cells of the peptides according to the invention. As anillustrative example, it should be mentioned the possibility of carryingout substitutions like, for example, leucine by valine or isoleucine,aspartic acid by glutamic acid, glutamine by asparagine, asparagine bylysine etc., it being understood that the reverse substitutions arepermitted in the same conditions. In some cases, it may also be possibleto replace a residue in the L-form by a residue in the D-form or thereplacement of the glutamine (Q) residue by a Pyro-glutamic acidcompound. The synthesis of peptides containing at least one residue inthe D-form is, for example, described by KOCH et al. (1977).

Tables 1 and 2 show the amino acid sequence of the preferred peptides ofthe invention (SEQ ID NOs:1–18) and, for each of these peptides, thecorresponding nucleotide sequence encoding these peptides (SEQ IDNOs:19–36; Table 2). However, since the genetic code is degenerated, itis clear that the nucleotide sequences given in Table 2 are, for each ofthese peptides, one specific example of the many possible examples ofsequence for coding these peptides. A person skilled in the art willeasily be capable of determining other nucleotide sequences coding forthe peptides of the present invention.

The peptides of the present invention may be prepared by any suitableprocess. Preferably, they are obtained by chemical synthesis in liquidor solid phase by successive couplings of the different amino acidresidues to be incorporated (from the N-terminal end to the C-terminalend in liquid phase, or from the C-terminal end to the N-terminal end insolid phase) wherein the N-terminal ends and the reactive side chainsare previously blocked by conventional groups. For solid phase synthesisthe technique described by Merrifield (J. Am. Chem. Soc., 85: 2149–2154)may be used. Alternatively, the technique described by Houbenweyl in1974 may also be used.

Typically, in order to produce a peptide chain using the Merrifieldprocess, a highly porous resin polymer is used, on which the firstC-terminal amino acid of the chain is fixed. This amino acid is fixed tothe resin by means of its carboxyl group and its amine function isprotected, for example, by the t-butyloxycarbonyl group. When the firstC-terminal amino acid is thus fixed to the resin, the protective groupis removed from the amine function by washing the resin with an acid. Ifthe protective group for the amine function is the t-butyloxycarbonylgroup, it may be eliminated by treating the resin with trifluoroaceticacid. The second amino acid which supplies the second residue of thedesired sequence is then coupled to the deprotected amine function ofthe first C-terminal amino acid fixed to the chain. Preferably, thecarboxyl function of this second amino acid is activated, for example,using dicyclohexylcarbodiimide, and the amine function is protected, forexample, using t-butyloxycarbonyl. In this way, the first part of thedesired peptide chain is obtained, which comprises two amino acids, theterminal amine function of which is protected. As before, the aminefunction is deprotected and the third residue can then be fixed, undersimilar conditions, to those used in the addition of the secondC-terminal amino acid. Thus, the amino acids which are to form thepeptide chain are fixed, one after another, to the amine group, which ispreviously deprotected each time, to the portion of the peptide chainalready formed, which is attached to the resin. When all the desiredpeptide chain is formed, the protecting groups are eliminated from thevarious amino acids which constitute the peptide chain and the peptideis detached from the resin, for example using hydrofluoric acid.

The peptides of the present invention may also be obtained by biologicalor genetic engineering processes. A typical example comprises the use ofexpression vectors comprising a polynucleotide sequence coding for thepeptide of interest (such vectors are within the scope of the presentinvention). Multimer of each peptide can also be produced by geneticengineering technology by expressing of a polynucleotide coding formultiple copies of a monomer, or coding for different monomers.

The peptides of the present invention may also be, incorporated inpolypeptides having a length varying from about 10 to about 50 aminoacids, preferably about 15 amino acids. According to a preferredembodiment, the peptides are incorporated in a tetrameric complex ofHLA-A0201 or HLA-B0702 comprising a plurality of identical or differentpeptides/polypeptides according to the invention. According to anotherpreferred embodiment, the peptides of the invention are incorporatedinto a support comprising at least two peptide molecules. Examples ofsuitable support include polymers, lipidic vesicles, microspheres, latexbeads, polystyrene beads, proteins and the like.

In another aspect, the invention is directed to a method for producing,in vitro, an immunogenic peptide, comprising: culturing in vitro, in asuitable culture medium, a cell incorporating an expression vector asdescribed previously; and collecting in the culture medium immunogenicpeptides produced by these cells. Therefore, the invention is alsoconcerned with cells, such as recombinant bacteria, transformed ortransfected by a virus or plasmid for expressing the peptides of theinvention. Methods for producing such cells and methods for using thesecells in the production of proteins/peptides are well known in the artand will no be described in detail herein.

The peptides, polypeptides and polynucleotides of the invention may alsobe used for producing polyclonal or monoclonal antibodies capable ofrecognizing and binding the same. Methods for producing such antibodiesare well known in the art. These antibodies may be used for thepreparation of a medicine for the prevention or treatment of human HIV-1infections.

D) Pharmaceutical Compositions

The peptides/polypeptides of the present invention, the polynucleotidescoding the same, and polyclonal or monoclonal antibodies producedaccording to the invention, may be used in many ways as antitumoralagents, for the preparation of pharmaceutical compositions, for thepreparation of an antitumoral vaccine, for the treatment or theprevention of HIV infections.

Therefore, in another aspect, the invention is directed topharmaceutical compositions comprising:

-   a) at least one component selected from the group consisting of:    -   an immunogenic peptide/polypeptide and/or a polynucleotide        and/or a fragment thereof and/or an antibody as defined        previously; and    -   specific CD8 T-cells primed against an immunogenic        peptide/polypeptide and/or a fragment thereof; and-   b) a pharmaceutically acceptable vehicle or carrier.

According to a preferred embodiment, the composition further comprisesat least one CD4 peptide. More preferably, the CD4 peptide is linked tothe HIV CD8 epitope. Even more preferably, the CD4 peptide is an HIV CD4epitope.

The compositions of the invention may be in a solid or liquid form or inany suitable form for a therapeutic use. They may be formulated for arapid or slow release of its components and may further comprisecompounds for stimulating/inhibiting the immune system. The compositionsof the invention may be prepared according to conventional methods knownin the art.

E) Stimulation of a Immune Response

In another aspect, the invention is directed to a method for priminghuman CD8 cells in vitro, comprising the steps of:

-   a) isolating HLA-B0702 lymphoid or myeloid cells from human subject;    and-   b) loading in vitro the cells isolated at step a) with at least one    immunogenic peptide/polypeptide and/or polynucleotide as defined    previously.

According to a preferred embodiment, the method further comprises thesteps of:

-   c) isolating CD8+ T-cells from the subject; and-   d) using the cells primed at step b) for priming in vitro the CD8+    T-cells isolated at step c).

In a further aspect, the invention is directed to a method forstimulating a subject immune response comprising:

-   -   administering into a compatible subject HLA-B0702 lymphoid or        myeloid cells primed in vitro using the method defined        hereinabove; and/or    -   administering into a compatible subject CD8+ T-cells primed        according to the method using the method defined hereinabove.

The invention also provides an ex vivo stimulation method of the humanimmune response. This method comprises the steps of isolating from ahuman autologous lymphoid or myeloid cells; incubating these isolatedcells in vitro with at least one immunogenic peptide/polypeptide and ora polynucleotide as defined previously, these cells allowing theinduction of a cytotoxic response in vitro. In a related aspect, theinvention provides a method for stimulating in vivo a human immuneresponse against HIV-1, the method comprising administering to aHLA-B0702 patient in need thereof either cytotoxic cells, lymphoid ormyeloid cells treated in vitro according to the ex vivo stimulationprocess of the invention.

In another aspect, the invention is directed to a method for detectingan early CTL response against HIV-1. This method comprises the steps of:

-   -   providing a tetrameric complex of HLA-B0702 comprising at least        one immunogenic peptide according to the invention;    -   incubating this complex with peripheral blood lymphocytes of a        subject; and    -   determining the presence of HIV-1 specific CTL.        The presence of HIV-1 specific CTL may be done by comparing        cells from the subject with normal cells in culture using any        suitable method such as FACS.

In another aspect, the invention is directed to a method for stimulatinga human immune response against HIV-1, comprising:

-   -   isolating from a human HIV-1 specific CTLs;    -   amplifying ex vivo the isolated HIV-1 specific CTLs; and    -   injecting in a compatible human in need thereof, the HIV-1        specific CTLs.

The following examples are illustrative of the wide range ofapplicability of the present invention and is not intended to limit itsscope. Modifications and variations can be made therein withoutdeparting from the spirit and scope of the invention. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice for testing of the present invention, thepreferred methods and materials are described.

EXAMPLE 1

Materials and Methods

HLA-B7B7K^(d) Chimerical Transgene and Animals.

The HLA-B*0702 gene was isolated from a cosmid library from theHLA-B*0702 homozygous HHK lymphoblastoïd human cell line. A 1.5 KbEcoRI-Kpn I fragment (promoter, exons 1 to 3) of the HLA-B*0702 gene wasligated to a 4.1 Kb Kpn I-Hind III fragment (exons 4 to 8) of theH-2K^(d) gene. The chimerical HLA-B7B7K^(d) gene was micro-injected asan EcoRI-Hind III fragment in C57BL/6×SJL oocytes. Transgenic animalswere backcrossed (×12) on C57BL/6 JICO(H-2^(b)) mice, before derivationof animals homozygous for the transgene. These mice were subsequentlyintercrossed with H-2 K^(bo) D^(bo) double KO mice (backcrossed 6 timeson C57BL/6 JICO, (7, 11) to derive HLA-B7B7K^(d), H-2 K^(bo)D^(bo)double KO homozygous mice (backcrossed 6 times on C57BL/6 JICO). Micewere bred in our animal facility and used for experimentation between 6and 10 weeks of age.

Peptide Binding

Peptides, purchased from SYNT:EM (Nimes, France), were dissolved in DMSO(1 mg of peptide/20 μl) and subsequently diluted in PBS (2 mg/ml).Peptides and HLA-B*0702 transfected TAP-T2 (7) cells were incubatedovernight at 37° C. (1×10⁶ cells/ml) in FCS-free medium supplementedwith 100 ng/ml of human β2-microglobulin (SIGMA, St Louis, Mo.) in theabsence (negative control) or presence of either reference humancytomegalovirus (CMV) pp65–265–274 (RPHERNGFTV, R10V, SEQ ID NO: 50) ortested peptides at various final concentrations (100, 10, 1 and 0.1 μM).Following a 1 h incubation with Brefeldin A (0.5 μg/ml, SIGMA),T2-B*0702 cells were labelled (30 min, 4° C.) with saturatingconcentration of ME.1 anti-HLA-B07 mAb, then washed twice and finallystained with FITC-conjugated F(ab)′2 goat anti-mouse Ig, before FACSanalysis. For each peptide, the concentration needed to reach 20% of themaximal fluorescence (as defined with the R10V peptide) was calculated.Relative affinity is the ratio of the concentrations of the tested andR10V reference peptide needed to reach this value: the lower therelative affinity, the stronger the binding.

Induction of CTL and Cytolytic Assays

For peptide immunisations, groups of 6 mice were injected s.c. at thebase of the tail with 50 μg of HLA-B0702-restricted peptide and 140 μgof the Ia^(b)-restricted helper peptide (hepatitis B virus core 128–140,TPPAYRPPNAPIL, T13L, SEQ ID NO: 51) (8) co-emulsified in 100 μl ofincomplete Freund adjuvant (IFA, Difco, Detroit, Mich.). Eight dayslater, spleen cells were re-stimulated in vitro as described before withpeptide-loaded, LPS-induced syngeneic lymphoblasts (9). On day 6,cultured cells were tested in a 4 h ⁵¹Cr-release assay, usingexperimental or control human CMV pp65–265–274 R10V peptide-pulsed,⁵¹Cr-labeled HLA-B7B7K^(d)-P815 cells. Specific lysis was calculated asfollow: (experimental−spontaneous release)/(total−spontaneousrelease)×100 subtracting the background lysis of R10Vcontrol-peptide-loaded target cells. Mice were considered as responderwhen specific lysis ≧10% was observed.

Immunofluorescence Assays.

Red cell-depleted, nylon-wool purified spleen T lymphocytes wereanalyzed for MHC class I molecule expression in an indirectimmunofluorescence assay. First layer mAb (B8-24-3 anti-H-2K^(b), B22.249.R19 anti-H-2 D^(b), and ME.1 anti-HLA-B0702) were incubated atsaturating concentrations (30 min, 4° C.) with cells. After 3 washes,mAb fixation was revealed with FITC-conjugated F(ab)′2 goat anti-mouseIg (Caltag, San Francisco, Calif.), and cells were FACS-analyzed (FACSCalibur, Beckton Dickinson, San Jose, Calif.). Percentages of CD4⁺ andCD8⁺ splenic T lymphocytes were determined by double staining usingFITC-conjugated rat anti-mouse CD4 (RM4-5) and phycoerythrin-conjugatedrat anti-mouse CD8-β (CT-CD8b) mAb (Caltag). Expression of the differentT cell receptor (TCR) Vβ chains was similarly analyzed using purifiedVβ2 (B.20.6), Vβ4 (KT.10.4), Vβ5.1,.2 (MR.9.4), Vβ6 (44.22.1), Vβ7(TR.130), Vβ8.1,.2,.3 (F.23.1), Vβ9 (MR.10.2) Vβ10 (B.21.5), Vβ11(RR.3.15), Vβ12 (MR.11.1), Vβ13 (MR.12.4), Vβ14 (14/2) and Vβ17(KJ.23.288.1) specific mAb. Fixation of these mAb was revealed withFITC-conjugated F(ab)′2 goat anti-mouse Ig (Caltag) and then CD8⁺ Tcells were labeled with phycoerythrin-conjugated rat anti-mouse CD8 mAb.Human HLA-B0702 phenotyping was performed on Ficoll (Pharmacia) purifiedPBL by indirect immunofluorescence as indicated above using ME.1anti-HLA-B7 antibody.

Human CTL In Vitro Restimulation and Cytolytic Assays.

Blood samples were obtained following written informed consent fromplatelet healthy donors tested serologically negative for HIV, HCV andHBV viruses. Nitrogen-frozen HLA-B0702+Ficoll-purified human peripheralblood mononuclear cells (PBMC) were thawed and incubated (4×10⁶/well) in24 well-plates in RPMI 1640, 1 mM sodium pyruvate, 100 IU/ml penicillin,100 μg/ml streptomycin, 10 mM hepes and non-essential aminoacids (allfrom GibcoBRL, Paisley, UK) supplemented with 10% human serum (InstitutJacques Boy, Reims, France). They were stimulated with influenza-derivedpeptides at 2×10⁻⁶ M. On day 3 recombinant human IL7 (25 ng/ml, kindlyprovided by Sanofi-Synthelabo, Labège, France) was added and on day 7,human IL2 was added at 10 IU/ml (Roche, Mannheim, Germany) with freshmedium. On day 16, CD8⁺ T cells were selected using CD8 Microbeads(Miltenyi Biotec, Bergish Gladbach, Germany). CTL lines weresubsequently restimulated twice monthly using peptide-pulsedEBV-transformed γ-irradiated (50 Gy) autologous cells. Cytolytic assayswere done on ⁵¹Cr-labeled peptide-pulsed HLA-B*0702-transfected TAP⁻ T2cells. Specific lysis was calculated as follow:(experimental−spontaneous release)/(total−spontaneous release)×100subtracting the background lysis (which never exceeded 5%) ofHLA-B0702-restricted, HIV1-derived, GP41 (843–851) IPRRIRQGL (SEQ ID NO:42) epitopic peptide-pulsed target cells (10).

Results

Results are shown hereinafter in Tables 1 and 2.

TABLE 1 Epitopic peptides derived from HIV proteins, presented by themolecule HLA-B7 of class I and inducing a CD8 cytotoxic cellularresponse (CTL) in transgenic mice B7B7K^(d+/+) H-2K^(−/−) H-2D^(−/−)Protein and epitopic Fixation SEQ ID peptides Test ⁵¹Cr release test**NO: N° Name Sequence RA* R/T Lysis (%) 1. Canonic peptides GAG  1 6342Y10LF YPLASLRSLF 3,25 1/6 37 ENV  2 6337 A10VV APTKAKRRVV 1,14 2/2 72;58  3 6339 R10LL RPVVSTQLLL 2,25 2/4 53; 50 VIF  4 6332 K10KL KPPLPSVTKL3,55 2/2 61; 31  5 7023 I10VI IPLGDARLVI 2,33 4/6 37; 37; 22; 22  6 7024H10HI HPRISSEVHI 3,70 6/6 20; 18; 51; 31; 70; 48  7 7025 S10EVSPHPRISSEV 1,11 6/6 18; 11; 38; 30; 49; 18  8 7020 T9PL TPKKIKPPL 1,295/6 46; 24; 18; 18; 11; 6 TAT  9 7019 P10QV PPQGSQTHQV 8,97 2/3 20; 13REV 10 7012 L9RL LPLPPLDRL 6,13 3/6 22; 12; 14 11 7026 L9TL LPPLDRLTL3,51 4/6 37; 20; 56; 23 VPU 12 7016 Q10AL QPIQIAIAAL 6,5 6/6 14; 40; 16;15; 13; 32 2. Non-canonic peptides, called ARFP (alternative readingframe peptides) POL 13 7029 A9RL AAISPVLRL (15) 1/6 17 14 7028 S10PVSPVLRLRPPV 0,85 2/6 24; 46 ENV 15 7033 Q10QM QPPLYFVHQM (49) 2/6 15; 1316 7034 G10QT GPHMPVYPQT (22) 1/5 10 17 7031 M9PT MPVYPQPT 0,94 3/6 17;28; 19 GAG 18 7035 Q9VF QPRSDTHVF 1,56 6/6 61; 38; 55; 48; 43; 33*Represents the ratio, between the concentrations of the peptide testedand the reference peptide (R10V human CMV pp65 265–274), that isnecessary to reach 20% of the maximal level of HLA-B*0702 moleculescapable of being stabilised at the surface of T2 cells in presence of asaturant concentration of the reference peptide. **Number of micepresenting a CTL response specific to the peptide under analysis (⁵¹Crrelease test) with respect to the total number of mice tested. The lysispercentage of these mice is given for a ratio (effector:target) of 30:1.

TABLE 2 Epitopic peptides derived from HIV proteins and preferrednucleic sequences coding the same. SEQ ID NO: 1. Canonic PeptidesGAG: 1. Y10LF Y   P   L   A   S   L   R   S   L   F 1 B.-.NL43E9 * TATCCT TTA GCT TCC CTC AGA TCA CTC TTT 19 ENV: 2. A10VVA   P   T   K   A   K   R   R   V   V 2 BRU 7306–7335 GCA CCC ACC AAGGCA AAG AGA AGA GTG GTG 20 3. R10LLR   P   V   V   S   T   Q   L   L   L 3 BRU 6571–6600 AGG CCA GTA GTATCA ACT CAA CTG CTG TTG 21 VIF: 4. K10KLK   P   P   L   P   S   V   T   K   L 4 BRU 5100–5129 AAG CCA CCT TTGCCT AGT GTT ACG AAA CTG 22 5. I10VII   P   L   G   D   A   R   L   V   I 5 BRU 4791–4820 ATC CCA CTA GGGGAT GCT AGA TTG GTA ATA 23 6. H10HIH   P   R   I   S   S   E   V   H   I 6 BRU 4764–4793 CAT CCA AGA ATAAGT TCA GAA GTA CAC ATC 24 7. S10EVS   P   H   P   R   I   S   S   E   V 7 BRU 4758–4787 AGC CCT CAT CCAAGA ATA AGT TCA GAA GTA 25 8. T9PL T   P   K   K   I   K   P   P   L 8BRU 5085–5111 ACA CCA AAA AAG ATA AAG CCA CCT TTG 26 TAT: 9. P10QVP   P   Q   G   S   Q   T   H   Q   V 9 BRU 5583–5612 CCT CCT CAA GGCAGT CAG ACT CAT CAA GTT 27 REV: 10. L9RLL   P   L   P   P   L   D   R   L 10 B.US.WEAU160 CTT CCT CTA CCA CCGCTT GAT CGA CTT 28 11. L9TL L   P   P   L   D   R   L   T   L 11B.US.WEAU160 CTA CCA CCG CTT GAT CGA CTT ACT CTT 29 VPU: 12. Q10ALQ   P   I   Q   I   A   I   A   A   L 12 BRU 5646–5675 CAA CCT ATA CAAATA GCA ATA GCA GCA TTA 30 2. Non-canonic peptides, called ARFP(alternative reading frame peptides) POL 1. A9RLA   A   I   S   P   V   L   R   L 13 BRU 4167–4190 GCA GCA ATT TCA CCAGTA CTA CGG TTA 31 2. S10PV S   P   V   L   R   L   R   P   P   V 14 BRU4173–4202 TCA CCA GTA CTA CGG TTA AGG CCG CCT GTT 32 ENV 3. Q10QMQ   P   P   L   Y   F   V   H   Q   M 15 BRU 5945–5974 CAA CCA CCA CTCTAT TTT GTG CAT CAG ATG 33 4. G10QTG   P   H   M   P   V   Y   P   Q   T 16 BRU 6008–6037 GGG CCA CAC ATGCCT GTG TAC CCA CAG ACC 34 5. M9PT M   P   V   Y   P   Q   T   P   T 17BRU 6017–6043 ATG CCT GTG TAC CCA CAG ACC CCA ACC 35 GAG 6. Q9VFQ   P   R   S   D   T   H   V   F 18 BRU 827–853 CAG CCC AGA AGT GAT ACCCAT GTT TTC 36

EXAMPLE 2

Example 2 relates to the validation of the peptides of the presentinvention from HIV⁺ patient. A first set of experiments were done by theinventors with frozen PBMCs (peripheral blood mononuclear cells) fromHIV⁺ patients. No clear response was observed. However, these sameexperiments were successfully achieved with fresh blood samples fromHIV⁺ patients. Here is a summary of the results.

Nine HIV⁺ HLA-B7⁺ patient samples were identified by serological typing.After a thirty-hours peptide stimulation, each sample was tested ex vivofor IFNγ secretion by ELISpot assay. Statistical analysis of all 9patient samples revealed that:

-   i) the eighteen selected peptides, as well as the peptides described    in the litterature, elicited responses;-   ii) response profiles are variable among peptides, both new and    already known, as well as among patients;-   iii) five peptides seem particularly immunogenic in vivo and will be    incorporated into the polyepitopic construct;

Two individuals (one HLA-B7⁺ HIV⁻ and the other HLA-B7³¹ HIV⁺) wereincluded in this example and showed no IFNγ ex vivo response followingexposure to these peptides.

Fourteen peptides are thus interesting for a future polyepitopicapproach. In the case of two patients, it was observed that:

-   i) a depletion of CD4⁺ cells from PBMC harvests does not modify the    observed IFNγ profile,-   ii) a cytolytic response can be elicited after two courses of in    vitro stimulation (as tested by ⁵¹Cr release assay).    Results

Results are shown in FIGS. 1 to 6 and in Tables 3 and 4.

As it can be appreciated, FIG. 1 shows the BNC's pattern for IFNγELISPOT responses against HIV-1 derived epitopic (known and new “coding”peptides), and more specifically shows the mean number (duplicates) ofspecific spots for 4.10⁵, 2.10⁵, and 10⁵ PBMCs.

BNC HIV-1⁺ patient blood was ficolled and PBMCs were directly stimulatedby each peptide (5 ug/ml) or by an irrelevant peptide, such as G9AT.Elispot assay was revealed 36 h later. The immunodominant HLA-B7restricted CMV peptide (T10M) was used for control. It will beunderstood that the methods or techniques used in Example 2 are wellknown, thus there is no need to further described them.

FIG. 2 shows the ELISPOT assay on human HIV⁺ PBMC (see FIG. 1 fortechnique). In this Figure, each spot corresponds to the number ofspecific spots per million of PBMCs of one HIV⁺ patient. 9 HIV⁺ patientswere tested and 2 human controls were included in this study (one HIV⁺HLA-B7⁻ and the other one HIV⁻ HLA-B7⁺). For each patient, ELISPOT assayresponse is positive in case of a the number of spots=mean ofbackground+3 SD. Positive responses for all new “coding” peptides wereobserved. Nevertheless, responses for the L9TL peptide don't appearsignificant.

NB: CD4⁺ cell depletion of PBMCs has been done with 3 HIV⁺ patients: nodifference in term of ELISPOT responses was observed, implying a role ofCD8⁺ cells in IFNγ release after peptide stimulation.

FIG. 3 shows an ELISPOT assay on human HIV+ PBMCs with ARFP peptides(see FIGS. 1 and 2 for technique).

Among 9 HIV⁺ patients, positive responses were observed for all ARFPpeptides. Globally, for each patient, 3 or 4 ARFP peptides areimmunogenic. Despite a lower intensity of responses comparatively to“coding” peptides, it was observed that all 6 peptides are immunogenic.

FIG. 4 shows the BNC's pattern for ⁵¹Cr release assay against predictedHIV-1 derived “coding” epitopic peptides. In this study, BNC HIV-1⁺patient blood was ficolled and PBMCs were stimulated 2 times bypeptide-pulsed autologous PHA blasts. ⁵¹Cr release assay was done onpeptide-pulsed T2-B7 cells. Non specific lysis was detected by acalpaine peptide. Non HIV peptides were used as controls: T10M (CMV) andG9AT (HCV). As it can be appreciated, the ⁵¹Cr release assay in thisstudy has confirmed results obtained by ELISPOT assay. It has beenpossible to do this experiment with PBMCs of an other HIV⁺ patient.ELISPOT responses were also confimed by ⁵¹Cr release assay.

FIG. 5 shows the BNC's and TAH's pattern for ⁵¹Cr release assay againstpredicted HIV-1 derived ARFP epitopic peptides (see FIG. 4 fortechnique). As it can be demonstrated in this study, patterns of ⁵¹Crrelease assay responses for two HIV⁺ patients (BNC and TAH) were similarto those observed with ELISPOT assay. Therefore, this study shows thatthe 6 ARFP peptides stimulate CTL responses in human.

FIG. 6A shows IFNγ intracellular labeling with TAH PBMCs. In this study,TAH HIV⁺ PBMCs were stimulated ex vivo during 12 hours with each ARFPpeptide and then, IFNγ intracellular labelling has been done. The A9RLpeptide was not tested. Similarities with ELISPOT assay responses wereobserved. Moreover, only CD8⁺ cells were IFNγ labelled, excluding thepossibility of a CD4⁺ cell stimulation by these peptides.

FIG. 6B shows IL4 intracellular labeling with TAH PBMCs (see FIG. 6A fortechnique). In this study, no IL4 production was detectable afterpeptide stimulation, neither for CD8⁺ cells, nor CD4⁺ cells. IL4 is acytokine produced during Th2 response. Thus, stimulation with peptidesof the present invention seems inducing Th1 response, implying astimulation of CTLs rather than helper T cells.

TABLE 3 list of “coding” HIV-1 epitopic peptides. Conservation* SEQ IDNO: Name Sequence Ref Isolate HXB2 location Clade B All clades 1. KnownHIV-1 derived epitopic peptides 37 S9WV SPRTLNAWV [1] LAI p24 (16–24)94% 90% 38 T9ML TPQDLNTML [1] LAI (48–56) 94% 66% 39 G9VL GPGHKARVL [3]SF2 B (223–231) 74% 51% 1 Y10LF YPLASLRSLF [4] — pr55 (484–493) 40% 12%40 K10LC KPCVKLTPLC [4] — Env (117–126) 87% 82% 41 R10SI RPNNNTRKSI [1]LAI gp160 (298–307) 38% 24% 42 I9GL IPRRIRQGL [1] LAI (843–851) 32% 33%43 F10LR FPVTPQVPLR [1] LAI Nef (68–77) 6% 4% 44 R9AL RPMTYKAAL [1] LAI(77–85) 6% 4% 45 T10PL TPGPGVRYPL [1] LAI (128–137) 17% 16% 46 T8RYTPGPGVRY [5] LAI (128–135) 15% 16% 47 Y9CY YPLTFGWCY [5] LAI (135–143)6% 4% 48 D10WK DPEKEVLQWK [4] — (175–184) 6% 4% 49 F9GL FPRIWLHGL [3]SF2 B Vpr (34–42) 21% 11% 2. Predicted HIV-1 derived epitopic peptidesa. Early proteins 9 P10QV PPQGSQTHQV [6] LAI Tat (58–67) 3% 1% 10 L9RLLPLPPLDRL [6] B.US.WEAU160 Rev (73–81) 3% 2% 11 L9TL LPPLDRLTL [6]B.US.WEAU160 (75–83) 3% 2% b. Structural proteins 2 A10VV APTKAKRRVV [4]— gp160 (497–506) 72% 40% 3 R10LL RPVVSTQLLL [4] — (252–261) 85% 41% c.Accessory proteins 4 K10KL KPPLPSVTKL [4] — Vif (160–169) 35% 22% 5I10VI IPLGDARLVI [6] LAI (57–66) 26% 17% 6 H10HI HPRISSEVHI [6] LAI(48–57) 47% 30% 7 S10EV SPHPRISSEV [6] LAI (46–55) 5% 3% 8 T9PLTPKKIKPPL [6] LAI (155–163) 37% 25% 12 Q10AL QPIQIAIAAL [6] LAI Vpu(2–12) <1% <1% *% of HIV strains presenting the same peptide amino-acidsequence (HIV databases, Los Alamos National Laboratory & NationalInstitutes of Health,http://hiv-web.lanl.gov/cgi-bin/EPILIGN/epilign.cgi).Comments:

-   1. S9WV, T9ML, R10SI, 19GL, F10LR, T10PL :C. Brander notes these are    B*0702 epitopes (HIV databases 2002).-   2. GPGHKARVL (SEQ ID NO: 39) was already described in HIV database    1998 ([2]). It has also been found as an immunodominant epitopic    peptide ([3])-   3. H10HI has been previously described as immunogenic in HIV-1    infected individuals with a less frequently sequence: HPRISSEVHI    (SEQ ID NO: 6) ([3])-   4. T9ML was described as an immunodominant epitopic peptide with the    decamere sequence TPQDLNTML (SEQ ID NO: 38) ([4]).-   5. K10LC, Y10LF and D10WK were described as subdominant peptides    ([4]).-   6. A10VV, R10LL, and K10KL were not described as immunogenic ([4]).

TABLE 4 list of “ARFP” new HIV-1 epitopic peptides (LAI strain) SEQ IDNO: Name Sequence POL 13 A9RL AAISPVLRL 14 S10PV SPVLRLRPPV ENV 15 Q10QMQPPLYFVHQM 16 G10QT GPHMPVYPQT 17 M9PT MPVYPQTPT GAG 18 Q9VF QPRSDTHVFReferences:

-   [1] Brander C., and Goulder P. The evolving field of HIV CTL epitope    mapping: new approaches to the identification of novel epitopes. HIV    molecular Immunology Database: IV-1, 2001.-   [2] Korber B., Brander C., Haynes C., Koup R., Moore J., and    Walker B. HIV molecular Immunology Database: I-B-11, 1998.-   [3] Altfeld M., Addo M. M., Eldridge R. L., Yu X. G., Thomas S.,    Khatri A., Strick D., Phillips M. M., Cohen G. B., Islam S. A.,    Kalams S. A., Brander C., Goulder P. J., Rosenberg E. S., Walker B.    D., and the HIV Study Collaboration. Vpr is preferentially targeted    by CTL during HIV-1 infection. J Immunol, 167(5):2743–52, Sep.    2001.4-   [4] Jin X., Roberts C. G., nixon D. F., Safrit J. T., Zhang L. Q.,    Huang Y. X., Bhardwaj N., Jesdale B., deGroot A. S., and Koup R. A.    identification of subdominant cytotoxic T lymphocyte epitopes    encoded by autologous HIV type 1 sequences, using dendritic cell    stimulation and computer-driven algorithm. AIDS Res Hum    Retroviruses, 16(1):67–76. Jan. 1, 2000-   [5] Lucchiari-Hartz M., van Endert P. M., Lauvau G., Maier R.,    Meyerhans A., Mann D., Eichmann K., and Niedermann G. Cytotoxic T    lymphocyte epitopes of HIV-1 nef: generation of multiple definitive    major histoccomptability complex class I ligands by proteasomes. J    Exp Med, 191(2): 239–52, Jan. 17, 2000.-   [6] Wain-Hobson S., Sonigo P., Danos O., Cole S., and Alizon M.    Nucleotide sequence of the IADS Virus, LAV. Cell, 40:9–17, 1985.-   [7] Smith, K. D. and Lutz, C. T. 1996. Peptide-dependent expression    of HLA-B7 on antigen processing-deficient T2 cells. J Immunol    156:3755.-   [8] Milich, D. R., Hughes, J. L., McLachlan, A., Thornton, G. B.,    and Moriarty, A. 1988. Hepatitis B synthetic immunogen comprised of    nucleocapsid T-cell sites and an envelope B-cell epitope. Proc Natl    Acad Sci USA 85:1610.-   [9] Firat, H., Garcia-Pons, F., Tourdot, S., Pascolo, S., Scardino,    A., Garcia, Z., Michel, M. L., Jack, R. W., Jung, G., Kosmatopoulos,    K., Mateo, L., Suhrbier, A., Lemonnier, F. A., and    Langlade-Demoyen, P. 1999. H-2 class I knockout, HLA-A2.1-transgenic    mice: a versatile animal model for preclinical evaluation of    antitumor-immunotherapeutic strategies. Eur J Immunol 29:3112.-   [10] Bette Korber, C. B., Barton Haynes, Richard Koup, John Moore,    Bruce Walker Eds. 1997. HIV Molecular Immunology Database. In.    Theoretical Biology and Biophysics Group, Los Alamos National    Laboratory, Los Alamos, N. Mex.

While several embodiments of the invention have been described, it willbe understood that the present invention is capable of, furthermodifications, and this application is intended to cover any variations,uses, or adaptations of the invention, following in general theprinciples of the invention and including such departures from thepresent disclosure as to come within knowledge or customary practice inthe art to which the invention pertains, and as may be applied to theessential features hereinbefore set forth and falling within the scopeof the invention or the limits of the appended claims.

1. An immunogenic peptide consisting of SEQ ID NO:
 12. 2. A compositioncomprising: a) an immunogenic peptide according to claim 1; and b) apharmaceutically acceptable vehicle or carrier.